Our laboratory has recently shown that amphetamines trigger the internalization of the dopamine transporter (DAT) by a series of intracellular events that are distinct from the generally established actions of amphetamines to inhibit dopamine (DA) uptake or to increase DA efflux. We have found that when applied to cell lines, cultured DA neurons or midbrain slices, amphetamine activates the small GTPases, RhoA and Rac-1 and triggers internalization of DAT by a specialized internalization pathway that requires the activation of the small GTPase, RhoA. Intriguingly, amphetamine must be transported into the cell to have these effects and its actions are actually blocked by cocaine, a drug that inhibits DAT and prevents amphetamine entry. We have also found that elevation of cAMP, via DA receptors or by amphetamine-induced adenylate cyclase activation, inactivates RhoA and serves as a break on carrier internalization, thus demonstrating an important interaction between PKA- and RhoA-dependent signaling in mediating the actions of amphetamines. These observations also imply the existence of a novel intracellular target for amphetamines and suggest new cellular pathways to target in order to disrupt amphetamine action. We are examining the role of a trace amine receptor (TAAR1) found in dopamine neurons, which may serve as a direct intracellular target for amphetamines. In addition, we have observed that the same amphetamine-activated RhoA-dependent mechanism also downregulates a glutamate transporter, EAAT3, present on DA neurons. We have identified the EAAT3 amino acid sequence responsible for this regulation, generated a cell-permeant fusion protein that interferes with the process and have used it explore the effects of amphetamine on excitatory neurotransmission in brain slices. These findings provide a new context in which to consider the actions of amphetamine on dopaminergic and glutamatergic signaling, and should provide insight into the unique neurotoxic and behavioral properties of this class of psychostimulant drugs. Glutamate transporters (also known as excitatory amino acid transporters or EAATs) present at the surface of neurons and supporting glial cells regulate the extracellular concentration of glutamate, the major excitatory neurotransmitter in the brain. By transporting glutamate back into the cell, these carrier proteins prevent glutamate from reaching toxic levels and also limit the extent and duration of transmitter signaling during glutamatergic neurotransmission. These carrier have an additional function in that they possess an anion channel activity that can regulate cellular excitability, which enables them to serve as sensors of glutamate levels outside the cell. Our laboratory has used site-directed mutagenesis, sulfhydryl modification, and chemical cross-linking approaches together with biochemical, and electrophysiological analyses of the mammalian carriers to examine the structural domains required for substrate transport and ion permeation. Using cysteine crosslinking studies and computational simulations we have identified several large-scale collective motions that are intrinsic to glutamate transporter trimers. Single cysteine residues introduced into the extracellular gate on each of the three subunits form reversible intersubunit crosslinks spontaneously and/or catalyzed by oxidizing reagents, such as copper phenanthroline. After crosslinking, substrate uptake, but not the substrate-activated anion conductance, is completely inhibited in these mutants. We have also developed new approaches that use chemical modifications of cytoplasmic cysteine residues to capture inward-facing conformations of these transporters. This work has shown that inward facing conformations of the carriers also mediate the anion channel activity associated with glutamate transporters. Overall, these results shed light on how the structure and conformation of these transporter proteins determine their functional dynamics and regulatory properties. Linkage analyses have revealed a significant association between the gene for the neuronal glutamate transporter, EAAT3 and Obsessive Compulsive Disorder (OCD). The majority of EAAT3 single nucleotide polymorphisms (SNPs) identified in OCD patient samples are synonymous coding SNPs or silent mutations, however, at least one non-synonymous coding variant in a highly conserved region of the carrier has been identified (T164A) and appears to display an altered function that cannot be simply explained by a reduced cell surface expression. Using electrophysiological techniques, radiolabeled glutamate uptake assays and surface expression assays, we have begun to characterize this mutation in greater detail in an effort to learn whether the alterations in the structural and functional properties EAAT3 have any role in the development of OCD.